Effects of biochar, zeolite and mycorrhiza inoculation on soil properties, heavy metal availability and cowpea growth in a multi-contaminated soil

Heavy metal pollution of agricultural soil has become a major serious concern. The development of suitable control and remediation strategies for heavy metal contaminated soil has become critical. The outdoor pot experiment was conducted to investigate the effect of biochar, zeolite, and mycorrhiza on the bioavailability reduction of heavy metals and its subsequent effects on soil properties and bioaccumulation in plants as well as the growth of cowpea grown in highly polluted soil. Zeolite, biochar, mycorrhiza, zeolite with mycorrhiza, biochar with mycorrhiza, and soil without any modifications were the six treatments used. The experiment was conducted with a completely randomized design and four replications. The results indicated that the combination of biochar with mycorrhiza had the highest values of root and shoot dry weight and the lowest heavy metal concentrations in root and shoot as well as bioconcentration and translocation factors for all heavy metals. The highest significant reductions in the availability of heavy metals over the control were found with biochar with mycorrhiza, which were 59.1%, 44.3%, 38.0%, 69.7%, 77.8%, 77.2% and 73.6% for Cd, Co, Cr, Cu, Ni, Pb and Zn, respectively. The application of biochar and zeolite either alone or in combination with mycorrhiza increased significantly soil pH and EC compared to mycorrhiza treatment and untreated soil. It can be concluded that the combination of biochar and mycorrhizal inoculation has great potential as a cost-effective and environmentally technique for enhancing heavy metal immobilization, lowering heavy metal availability and plant uptake, and improving cowpea plant growth.

. Effect of soil amendments and mycorrhiza on root and shoot dry weight of cowpea plant grown in contaminated soil. Error bars are the standard deviation. (Different letters above bars represent significant differences at P < 0.05 among various treatments. Bars with the same letters represent no significant difference).

Heavy metal concentrations in cowpea plants.
The changes in both root and shoot heavy metal concentrations of the seven heavy metals under different treatments are shown in Table 1. The highest concentrations were recorded for plants grown in untreated soil. In contrast, the lowest concentrations were found in the treatment of biochar followed by zeolite, especially in the presence of mycorrhiza. Bioconcentration factor (BCF). The BCF of the seven heavy metals under the different treatments of soil amendments and mycorrhiza are shown in Table 2. All treatments had significant effects on BCF compared to the untreated soil. The lowest effects were recorded with the application of biochar with mycorrhiza followed by the combination of zeolite with mycorrhiza for all heavy metals. Soil properties after harvest. The application of biochar and zeolite either alone or in combination with mycorrhiza increased significantly soil pH compared to mycorrhiza treatment and untreated soil (Fig. 3), which their soil pH was moderately alkaline (pH = 7.80). All applications increased significantly EC compared to untreated soil. When the biochar and zeolite additions were applied alone, soil pH and EC were significantly increased compared with those observed after mycorrhiza inoculation with them. The highest significant increases over the control were found with zeolite treatment, which were 8.31 and 1.98 dS m -1 for soil pH and EC, respectively.

Discussion
The efficiency of biochar, zeolite, and mycorrhiza in reducing heavy metal bioavailability in soil and bioaccumulation in plants, as well as in promoting the growth of cowpea grown in highly polluted soil, was investigated in the current study. The reduced plant biomass from the control treatment ( Fig. 1) must be explained by the Table 3. Effect of soil amendments and mycorrhiza on translocation factor (TF) of cowpea plants grown in contaminated soil. Means followed with similar letters within the same column are not different significantly at P < 0.05 level of probability based on LSD test.  Table 1) that caused plant toxicity and resulted in a decrease in biomass production 6 . Nawab et al. 18 found that the root and shoot growth of pea plants decreased with the high toxicity of Cr, Ni, Zn, Pb, and Cd in soil. The present study found that mycorrhiza can boost shoot biomass and adding biochar and zeolite to the soil can promote it (Fig. 1). The stimulating dry biomass by application of biochar and zeolite in combination with mycorrhiza may be attributed to the important role of biochar, zeolite, and mycorrhiza in lowering heavy metal bioavailability ( Fig. 2) resulting in lower plant uptake and phytotoxicity of heavy metals, which led to improved plant growth ( Fig. 1). This finding was in line with the results of prior studies on biochar 6 , zeolite 19 and mycorrhiza 20 when they were used to remediate soil that had been polluted by toxic metals. Biochar and zeolite boost plant growth during the remediation process by a variety of mechanisms, including toxic metal adsorption, higher soil water retention, encouraging plant growth-promoting microorganisms, and increased root development 19,21 . In our study, zeolite was found to be less effective than biochar as a soil amendment, despite a converse result being found when lower addition rates (0.5%) were used 19 . The increases in dry biomass as a result of biochar and zeolite treatment can be explained by increased nutrient availability and uptake in the soil and/or plant 22,23 and the improvement in soil physio-chemical properties 3,24 .
Moreover, Bashir et al. 25 reported that biochar improved nodule formation in mungbean plants, microbial activity in the soil, and increased shoot fresh biomass. Increased mycorrhizal colonization in the soil causes an increase in plant biomass 26 . Mycorrhiza help plants absorb water and nutrients by forming a pervasive hyphal network, increasing the efficiency of chemical fertilizers 27 . In metal-contaminated soils, mycorrhiza has been found in association with plant roots and has been suggested to protect plants against heavy metal toxicity and improve plant development 28 . Plants grown in heavy metal-contaminated soils can benefit from mycorrhiza by facilitating nutrient uptake, protecting them from metal toxicity, sequestering HMs into mycorrhiza structures, and improving phytostabilization 14,23 On the other hand, using biochar in combination with other amendments, such as soil microorganisms, can boost biochar's trace metal remediation capacity and improve crop growth 29 . The stimulative effect of biochar and mycorrhiza combined treatment could be attributed to the benefits of biochar to mycorrhiza as a source of reduced carbon compounds and available nutrients 12,23 .
Biochar amendment increases the physicochemical properties of soils, making them more suitable for mycorrhiza colonization by promoting spore germination, hyphal branching, and mycorrhiza growth 30,31 . Besides, biochar absorbs the substances that are hazardous to mycorrhizal fungus, effectively lowering the toxicity of many heavy metals 32 . Guo and Li 32 found that the combination of biochar and mycorrhiza protected the plants from heavy metal toxicity and promoted plant growth and the effect of their combined inoculation were greater than a single one.
The heavy metal concentrations in roots and shoots of the plants grown under biochar or zeolite combined with mycorrhiza inoculation applications were lower than in single applications, which were related to the interaction of biochar or zeolite with mycorrhiza. These significant reductions could be attributed to enhanced immobilization of available heavy metals in soil (Fig. 2), as well as a diluting impact due to increased plant biomass (Fig. 1). Mycorrhiza reduces heavy metal concentrations in roots and shoots, such as Cd, Cu, Mn, and Zn 26 . Mycorrhiza's efficiency to reduce Cd uptake increased after zeolite addition to the soil 15 . The application of biochar with mycorrhiza in heavy metal-contaminated soils reduced significantly heavy metal levels in the shoots by reducing heavy metal translocation from the roots 32 . www.nature.com/scientificreports/ The bioaccumulation factor results indicate that all treatments of soil amendments and mycorrhiza inhibited the bioaccumulation of selected heavy metals in cowpea ( Table 2). The accumulation of heavy metals in cowpea root and shoot tissues was smaller than the accumulation in soil, as evidenced by the fact that none of the BCF values exceeded one. The lower BCF values were most likely due to biochar and mycorrhiza's higher efficiency in reducing heavy metal uptake (Table 1). In general, adding biochar or mycorrhiza to soil reduced the bioavailable concentration of heavy metals in the soil, resulting in lower root uptake 6,33 . Metals adsorb on the surfaces of biochar and mycorrhiza, reducing their concentrations and mobilization in the soil, as well as their translocation in plants 34,35 . Moreover, biochar increase the pH, organic carbon content, and cation exchange capacity of the soil leading to lower the phytoavailability of heavy metals 36,37 .
The decrease in TF values (Table 3) could be related to metal precipitation in the root tissues as presented in Table 1. In comparison to shoots, a major portion of the heavy metals was contained in the roots. The use of biochar in combination with mycorrhiza is a viable treatment for lowering TF levels. According to the findings of prior studies, the TF values of heavy metals from roots to shoots were significantly reduced after biochar was applied to the multi-metal contaminated soil 6,38 . Moreover, mycorrhiza is effective in reducing heavy metal transport from the root to the shoots 26 , because heavy metals can be retained in mycorrhizal structures 39 . Plantmycorrhiza symbiosis could enable selective transport mechanisms of both essential minerals and heavy metals 40 . Mixing biochar with mycorrhiza in metal-contaminated soils reduced significantly heavy metal translocation from the roots 23,32. The increase in soil pH and organic matter will promote heavy metal immobilization 41 , resulting in a reduction in the plant's heavy metal uptake 6,42 . Both zeolite and biochar supply alkalinity to the soil causing insoluble metals 36,43 . In this study, the pH value of soil is increased with the application of biochar and zeolite (Fig. 3), which reduced the content of available heavy metals in the soil after harvest (Fig. 2). The soil organic matter was raised when biochar was applied 44,45 . According to Chen et al. 42 average concentrations of Cd, Pb, Cu, and Zn in plant tissues are lowered in comparison with control by 38%, 39%, 25%, and 17%, respectively, since biochar has a high pH value and increases organic matter content in the soil. Several studies proved the biochar and zeolite role in reducing the availability of heavy metals in soil 6,36,37,46 . Both biochar and zeolite reduce metal solubility in soils by many mechanisms: (1) supplying alkalinity and consequent pH rise 32,43 (2) promoting sorption by surface complexation 36,45 , (3) increasing cation exchange retention 19 , (4) organic complexation 19,47 , and (5) reduction of hydraulic conductivity and pore size of soils 19 . Due to metal binding and complex formation, zeolite was effective in lowering heavy metal mobility 46 . The positive cations in zeolite can exchange with specific heavy metal cations in soil solutions, giving it a high capacity for cation exchange 46,48 . Biochar can absorb soil heavy metals by ion exchange process, complexation, precipitation functions, electrostatic attraction, or transforming metals from an inorganic form to an organic form that altered the pollutants' bioavailability and mobility 7 . On the other hand, mycorrhizal soil inoculation could be vital in immobilizing heavy metals in the soil and altering their availability to plants 13,27,28 .
Mycorrhiza acts as a physical barrier by sequestering the heavy metals into mycorrhizal structures and immobilizing heavy metals in plant roots 28 . On the surface of the fungal structure, positive charge particles such as amino acids, cysteine, glutathione and thiol groups adsorb and later reduce heavy metals from one form to another 35 . Also, mycorrhiza creates extracellular polymeric substances, which have carboxyl, phosphoric, amine and hydroxyl functional groups and have a high affinity for metal adsorption through chelation, surface precipitation, and ion exchange mechanisms 49 . The fungal structure is often much finer than roots, and it has a strong potential to chelate heavy metals and reduce their bioavailability in the rhizosphere through metal speciation 50,51 . Mycorrhiza decreases heavy metal toxicity in plants by keeping heavy metals in mycorrhizal structures such as the fungal mycelium and vesicles, where high quantities of heavy metals are concentrated, preventing their mobilization to aerial plant tissues 27 . Moreover, many studies have shown that combining mycorrhiza and biochar has a positive impact on lowering bioavailable heavy metals in soil 14,30,52 .
The increase in soil pH caused by biochar and zeolite applications is due to their high pH (Table 4). Alkalinity is supplied to the soil by both zeolite and biochar 43 . The increase in soil pH after the application of biochar alone or in combination with mycorrhiza may be related to the pH of biochar itself (Fig. 3). The increase in soil pH is www.nature.com/scientificreports/ due to the dissolution of carbonates and hydroxides mainly present in the applied biochar's ash friction 7,53 . In this respect, Guo and Li 32 found that mycorrhiza has no effect on soil pH. Zhuo et al. 30 found that soil treated with a combination of biochar and mycorrhiza had a considerable increase in pH. Moreover, various researchers reported that biochar and zeolite increase the soil EC value 54 .

Conclusions
Heavy metal pollution of agricultural soil causes severe threats to ecosystem sustainability and human health, and it has become a major serious concern. The development of suitable control and remediation strategies for heavy metal-contaminated soil has become critical. The results of the present study suggest that the combined application of biochar or zeolite with mycorrhiza in metal-contaminated soil can improve cowpea growth, decrease metal bioavailability and mobility, and reduce metal uptake. Biochar was shown to be more effective than zeolite. This avenue of study will help in the remediation of metal-contaminated soils, resulting in improved plant growth and lower bioconcentration and translocation factors in a cost-effective and environmental technique. More specialized field-scale experiments are necessary to evaluate the potential role of biochar and mycorrhiza in the remediation of heavy metal-contaminated soils and validate their practical application with a variety of plant and climate conditions.

Materials and methods
Soil, zeolite, biochar and mycorrhiza. The soil used in this study was collected from an agricultural field near Mansoura, Dakahlia Governorate, Egypt (31°25′16.1′′E, 31°03′07.8′′N), at a depth of 20 cm. A standard approach narrated in Ryan et al. 55 was used to examine the physio-chemical properties of the studied soil. The properties of this soil are presented in Table 5.
The zeolite clinoptilolite which a hydrated aluminosilicate mineral that contain alkali and alkaline earth metals (Na, K, Ca, and Mg) was used in this experiment. It was purchased from a company in Giza, Egypt. The zeolite was sieved at 0.2 mm before being used. The biochar was produced using dried woods taken from mature trees of mango as feedstocks. They were exposed to direct sunshine before being sliced into little 10 cm pieces. Dry wood chips were covered thoroughly with aluminum foil to provide an oxygen-limited condition and pyrolyzed at 500 °C for 5 h in muffle furnace (Magma Therm, MT-1200-10-B2, Turkey). Biochar was cooled to room temperature before being crushed using a stainless-steel mill and sieved with a 2 mm mesh size. Mycorrhiza fungi used in this experiment were obtained from Soil, Water and Environment Research Institute, ARC, Egypt. The mycorrhiza used is an endomycorrhizal biofertilizer with 250 spores in 1 g of the carrier. The properties of zeolite and biochar used in this study are shown in Table 4.
Pot experiment. The outdoor pot experiment was conducted at Mansoura Horticulture Research Station, Dakahlia Governorate, Egypt, during the summer season of 2021. The experiment was conducted with a completely randomized design in four replications. The following six treatments were applied: zeolite, biochar, mycorrhiza, mixtures of zeolite and mycorrhiza, mixtures of biochar and mycorrhiza, and control (soil without any modifications). Both biochar and zeolite were applied at a rate of 2% w/w and completely mixed with soil alone to obtain homogeneity.
To prepare the artificially polluted soil, all of the pots were contaminated with a solution of seven heavy metal salts (CdSO 4 .8H 2 O, CoCl 2 .6H 2 O, Cr 2 (SO 4 ) 3 , CuSO 4 5H 2 O, Ni-SO 4 6H 2 O, Pb(C 2 H 3 O 2 ) 2 and ZnSO 4 7H 2 O) at a concentration of 3.2, 10, 85, 50, 50, 70 and 130 mg kg -1 for Cd, Co, Cr, Cu, Ni, Pb and Zn, respectively. The water solution with toxic metals was thoroughly mixed into the soil to ensure homogeneity then the plastic pots with a capacity of 7 kg were filled with contaminated soil. The pots were left to equilibrate for three weeks before seed sowing. During this time, the moisture content of the soil was maintained at 65 percent of its field capacity. www.nature.com/scientificreports/ After that, on 6 June 2021, soil was inoculated with mycorrhiza. To this end, the mycorrhizal biofertilizer at the rate of 1.5 g soil kg -1 was placed in the pots at a depth of 3 cm from the soil surface. Then, ten seeds of cowpea cv. Cream 7 were sown per pot at 2 cm deep and irrigated with tap water. After a week of emergence, the seedlings were trimmed to four of uniform size in each pot. The water content was kept at the field capacity level. All pots were irrigated with NPK fertilizer solution at a level of 60-45-45 kg per hectare, respectively, after 7 days of sowing.
The roots and shoots of plants in each pot were picked separately after five weeks of growth and cleaned thoroughly with tap water followed by distilled water to eliminate dust and soil, and then their fresh weights were recorded. After drying them for 72 h at 70 °C, the dry weights were recorded, and the samples were ground into a fine powder and placed in paper bags for heavy metal analysis. After plant harvests, a soil sample was taken from each pot, air-dried, crushed, sieved through a 2 mm sieve, and stored for physical and chemical analysis.
Analysis and measurements. For particle-size analysis, the hydrometer method was used. Jackson's method 56 was used to assess soil pH, organic matter (OM) content, and electrical conductivity (EC). The elemental content of C (carbon) was determined using the Thermo Scientific Flash 2000 elemental analyzer. The total and available concentrations of Cd, Co, Cr, Cu, Ni, Pb and Zn in the extracts were determined using Thermo Scientific TM ICAPTM 7000 Plus Series ICP-OES Ammann 57 . The available heavy metals were extracted by 0.005 M DTPA at pH 7 according to Lindsay and Norvel 58 .
The bioconcentration factor (BCF) and translocation factor (TF) of each heavy metal were determined according to Padmavathiamma and Li 59 , to evaluate the heavy metal absorption and translocation. The ratio of metal concentration in plant tissues to metal concentration in the soil is known as BCF, while the ratio of metal concentration in the plant shoot to metal concentration in the plant root is known as TF.
Statistical analysis. The ANOVA technique was used to calculate statistical analyses by Costat computer software according to Snedecor and Cochran 60 . The Least Significant Difference (LSD) test was used to evaluate the differences between means. The significance of the difference was determined using the P = 0.05 value.
Research involving plants statement. This study was developed with commercial seeds, therefore nonexotic or at risk of extinction, under controlled conditions, meeting all institutional, national and international guidelines and legislation for cultivated plants.

Data availability
All data generated and/ or analyzed during this study are available from the corresponding author on reasonable request.